TGF-beta-MEDIATED OSTEOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS

ABSTRACT

Methods and compositions are provided for the culture of MSC to provide osteogenic progenitor cells.

INTRODUCTION

Multipotent stem cell populations found in adult tissues have been ofgreat interest because they serve as reservoirs for tissue renewal aftertrauma, disease, and aging. One important type of adult stem cell, knownas the mesenchymal stem cell (MSC), is derived from bone marrow. MSCsare maintained in a relative state of quiescence in vivo, but inresponse to a variety of physiological and pathological stimuli, arecapable of proliferating then differentiating into osteoblasts,chondrocytes, adipocytes, or hematopoiesis-supporting stromal cells.However, little is understood regarding the cellular or molecular eventsunderlying MSC fate decisions.

Transforming growth factor β (TGF-β) proteins play important roles inthe regulation of many developmental processes and maintenance of normaltissue homeostasis (Massague, J. (1998). Annu Rev Biochem 67: 753-91).TGF-β initiates signaling by binding and activating themembrane-anchored type II and type I receptor Serine/Threonine kinases,which subsequently phosphorylate the effectors Smad2/Smad3.Phosphorylated Smad2/Smad3 can then form complexes with Smad4 andtranslocate into the nucleus. Accumulation of Smads in the nucleusresults in the activation or repression of downstream target genes byrecruiting various transcriptional coactivators or corepressors(Derynck, R. (1998) Nature 393(6687): 737-9). The central regulation ofbone differentiation and formation is controlled by the transcriptionalactivity of Runx2 and TAZ. Essential during osteogenic differentiationof mesenchymal progenitor cells, homozygous deletion of Runx2 in miceresults in the complete absence of osteoblasts and bone (Komori et al.(1997) Cell 89(5): 755-64; Otto et al. (1997) Cell 89(5):765-71). TAZ, aWW domain-containing molecule, functions as a transcriptional modulatorto stimulate bone development while simultaneously blocking thedifferentiation of mesenchymal stem cells into fat. These developmentaleffects occur through direct interaction between TAZ and thetranscription factors Runx2 and PPARγ, resulting in transcriptionalenhancement and repression, respectively, of selective programs of geneexpression (Hong (2006) Cell Cycle 5(2):176-9; Hong et al. (2005)Science 309(5737):1074-8.

The transforming growth factor-β (TGF-β) pathway plays a key role in thebalance between stem cell renewal and differentiation and has beenimplicated in chondrogenesis. However, the role of TGF-β in osteogenicdifferentiation remains unclear. In the present study, we investigatedthe role of TGF-β1 in multipotent differentiation of mouse bonemarrow-derived MSCs (BMSCs) in vitro.

SUMMARY OF THE INVENTION

Compositions and methods are provided that relate to cultures for theosteogenic differentiation of mesenchymal stem cells. MSC are culturedin the presence of a TGB-β, including TGF-β1, or TGF-β3, in an amountsufficient to induce osteogenic differentiation. The osteogenicprogenitor cells thus derived may be isolated from the culture, orotherwise utilized for various purposes. For example, osteogenic cellsof the invention find use in therapeutic methods, e.g. fortransplantation as a source of osteogenic progenitors; and the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Isolation of bone marrow-derived MSCs and detection of Sca-1 inMSCs (A) Mesenchymal stem cells at passage 5 isolated from adult mousefemur bone marrow were attached to tissue culture plate 24 hours afterseeding. (B) Nuclear staining using DAPI for all bone marrow-derivedMSCs in the field. (C) Staining of same cells shown in B with anti-Sca-1antibody. Staining is consistent with membrane pattern, the expectedlocation of Sca-1.

FIG. 2. Osteogenic and adipogenic differentiation of MSC. (A) and (C)MSCs were cultured in regular maintain medium for 7 day. MSCs werecultured in osteogenic medium (B) and adipogenic medium (D) for 7 days.Alizarin red staining was performed on (A) and (B). Red Oil Staining wasperformed on (C) and (D).

FIG. 3. TGF-β1 enhanced expression of osteogenic genes and inhibitedexpression of adipogenic genes. Bone marrow-derived MSCs treated withTGF-β1 at 10 ng/ml for 2 weeks. RNA was collected from cells andreal-time PCR was used to exam the expression of stem cells markers (A),osteogenic genes (B), and adipogenic genes (C).

FIG. 4. Alkaline phosphatase activity at 2 weeks significantly increasedin bone marrow-derived MSCs treated with TGF-β1 at 10 ng/ml comparedwith untreated group. Alkaline Phosphatase (AP) Staining on MSCscultured for 2 weeks with (B) or without (A) 10 ng/ml of TGF-β1. (C)Alkaline phosphatase activity was measured on MSCs treated with TGF-β1at 10 ng/ml for 2 weeks.

FIG. 5. The expression of TGF-β1 signaling pathway on bonemarrow-derived MSCs. MSCs treated with TGF-β1 at 10 ng/ml for 2 weeks.RNA was collected from cells and real-time PCR was used to exam theexpression of TGF-β1, Smad-2, and Smad-3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Compositions and methods are provided that relate to cultures for theosteogenic differentiation of mesenchymal stem cells. MSC, which may bebone marrow derived (BMSC) or adipose tissue derived (AMSC) are culturedin the presence of TGB-β, including TGF-β1, or TGF-β3, in an amountsufficient to induce osteogenic differentiation, usually for at leastone week and not more than about 3 weeks. The resulting osteoblasts maybe implanted into injured bone to promote fracture healing. TGF-β1and/or -β3 may also be injected around the wound or intravenously toenhance mesenchymal progenitors at the repair site to differentiate intoosteoblasts, in order to accelerate the healing process. MSCs will moveto the site of injury using homing signals and contribute to boneregeneration at the site of bone injury.

There are two types of bone ossification, intramembranous andendochondral. The former gives rise to flat bones, especially skull andclavicle. Intramembranous ossification does not go through a step oflaying down provisional cartilage. MSCs may first convert intoosteochondral progenitors by downregulating adipogenic pathway throughTAZ upregulation via TGF-β1 or TGF-β3. Then, the specific hormone maypromote either intramembranous or endochondral differentiation. Thiscell dual capability is evidenced in FIG. 6, which shows that exposureto TGF-β1 or TGF-β3 resulted in osteoblast matrix deposition, asevidenced by positive alkaline phosphatase activity using culturedcells. In particular, TGF-β1 down-regulates chondrogenesis and increasesosteogenesis.

Advantages of the present invention include the use of a biologic agent,human recombinant TGF-β, which can serve as a therapeutic agent for bonehealing via osteogenic stimulation. This simple treatment isphysiological, unlike the artificial treatments such as dexamethasone,β-glycerophosphate and ascorbic acid that are currently employed forbone engineering. For example, the use of dexamethasone is problematicdue to the fact that steroids are known inhibitors of wound healing invivo.

The effect of TGF-β1 in fracture healing may be more effective thanother osteogenic cytokines, such as BMP-2 and BMP-4. TGF-β1 inperipheral blood dramatically increases at two weeks after trauma andcontinue at the high level at six months after trauma, while BMP-2 andBMP-4 in peripheral blood can not be detected during the whole healingprocess. Therefore, TGF-β offers a physiologically relevant stimulationto bone regeneration, unlike BMPs.

In other embodiments of the invention, adenoviral gene transfer isutilized as a safe and effective way to obtain high and stableexpression of protein. A recombinant adenovirus expressing one or bothof TGF-β1 and TGF-β3 may be generated and transferred into MSCs tostimulate osteogenesis in vitro.

In other embodiments, a biomaterials scaffold serves as a syntheticextracellular matrix (ECM) to organize cells into a three-dimensional(3D) architecture and to present stimuli, which direct the growth andformation of a desired tissue. Human MSCs are seeded on the desiredscaffolds and cultured with human recombination TGF-β1 or TGF-β3 or bothto generate osteoblasts in vitro. Scaffolds can be impregnated withrecombinant TGF-β1 or TGF-β3 so as to release growth factor over asustained period of time, e.g. for at least about 3 month 3 and up toabout 6 months stimulation of osteogenesis. For example, MSCs can beseeded onto TGF-β1 or TGF-β3 impregnated scaffolds and implanted into asite of bone injury. In some embodiments the seeded MSC are transfectedwith adenovirus directing expression of one or both of TGF-β1 andTGF-β3.

As an alternative method to in vitro culture, short sequence syntheticpeptides of TGF-β1 or TGF-β3, which contain the receptor binding motif,can be generated introduced into the region of a bone fracture, in orderto induce endogenous MSCs around the wound to differentiate intoosteoblasts.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., an autoimmune disease state,including prophylaxis, lessening in the severity or progression,remission, or cure thereof.

The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to alter a protein expressionprofile.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Mesenchymal Stem Cell (MSC). As used herein, the term MSC refers to acell capable of giving rise to differentiated cells in multiplemesenchymal lineages, specifically to osteoblasts, adipocytes, myoblastsand chondroblasts. Generally, mesenchymal stem cells also have one ormore of the following properties: an ability to undergo asynchronous, orsymmetric replication, that is where the two daughter cells afterdivision can have different phenotypes; extensive self-renewal capacity;and clonal regeneration of the tissue in which they exist, for example,the non-hematopoietic cells of bone marrow. “Progenitor cells” differfrom stem cells in that they typically do not have the extensiveself-renewal capacity. In contrast to previously reported MSC andmultipotent mesenchymal cell populations, the cells of the invention donot require lengthy time in culture prior to the appearance of the MSCphenotype, i.e. cells with the MSC phenotype and are responsive tocanonical wnt signaling pathways are present in freshly isolated orprimary cultures that have been cultured for less than about 20passages; usually less than about 10 passages.

MSC have been harvested from the supportive stroma of a variety oftissues. In both mouse and human a candidate population of cells hasbeen identified in subcutaneous adipose tissue (AMSC). These cells havedemonstrated the same in vitro differentiation capacity as BM-MSC forthe mesenchymal lineages, osteoblasts, chondrocytes, myocytes, neurons,and adipocytes (Zuk et al. (2002) Mol Biol Cell 13, 4279-95; Fujimura etal. (2005) Biochem Biophys Res Commun 333, 116-21). Additionally, cellsurface antigen profiling of these cells has revealed similar cellsurface marker characteristics as the more widely studied BM-MSC(Simmons et al. (1994) Prog Clin Biol Res 389, 271-80; and Gronthos etal. (2001) J Cell Physiol 189, 54-63).

MSC may be characterized by both the presence of markers associated withspecific epitopes identified by antibodies and the absence of certainmarkers as identified by the lack of binding of specific antibodies. MSCmay also be identified by functional assays both in vitro and in vivo,particularly assays relating to the ability of stem cells to give riseto multiple differentiated progeny; assays for responsiveness tocanonical wnt signaling; and the like.

The cells of interest are typically mammalian, where the term refers toany animal classified as a mammal, including humans, domestic and farmanimals, and zoo, laboratory, sports, or pet animals, such as dogs,horses, cats, cows, mice, rats, rabbits, etc. Preferably, the mammal ishuman.

The cells which are employed may be fresh, frozen, or have been subjectto prior culture. They may be fetal, neonate, adult. MSC may be obtainedfrom adipose tissue (see U.S. Patent application 20030082152); bonemarrow (Pittenger et al. (1999) Science 284(5411):143-147; Liechty etal. (2000) Nature Medicine 6:1282-1286); G-CSF or GM-CSF mobilizedperipheral blood (Tondreau et al. (2005) Stem Cells 23(8): 1105-1112),or any other conventional source.

In some embodiments, the homogeneous MSC composition is stable innon-differentiating culture conditions, where the proportion of cells inthe composition that have an MSC phenotype are maintained over multiplepassages. Such cells may be maintained for at least about two passages;at least about five passages; at least about ten passages; or more.

Osteogenic culture conditions. Differentiating cells are obtained byculturing or differentiating MSC in a growth environment that enrichesfor cells with the desired phenotype, e.g. osteoblasts, osteogenicprogenitor cells, etc. The culture may comprise agents that enhancedifferentiation to a specific lineage.

Osteogenic differentiation may be performed by plating cells andculturing to confluency, then culturing in medium comprising TGF-β1and/or TGF-β3 at a concentration of from around about 100 pg/ml toaround about 100 ng/ml, usually around about 10 ng/ml.

Following the differentiation in culture, the culture will usuallycomprise at least about 25% of the desired differentiated cells; moreusually at least about 50% differentiated cells cells; at least about75% differentiated cells, or more. The cells thus obtained may be useddirectly, or may be further isolated, e.g. in a negative selection toremove MSCs and other undifferentiated cells. Further enrichment for thedesired cell type may be obtained by selection for markerscharacteristic of the cells, e.g. by flow cytometry, magnetic beadseparation, panning, etc., as known in the art.

Analysis or separation by cell staining may use conventional methods, asknown in the art. Techniques providing accurate enumeration includeconfocal microscopy, fluorescence microscopy, fluorescence activatedcell sorters, which can have varying degrees of sophistication, such asmultiple color channels, low angle and obtuse light scattering detectingchannels, impedance channels, etc. The cells may be selected againstdead cells by employing dyes associated with dead cells (e.g. propidiumiodide).

The affinity reagents may be specific receptors or ligands for the cellsurface molecules indicated above. In addition to antibody reagents,polynucleotide probes specific for an mRNA of interest, peptide-MHCantigen and T cell receptor pairs may be used; peptide ligands andreceptor; effector and receptor molecules, and the like. Antibodies andT cell receptors may be monoclonal or polyclonal, and may be produced bytransgenic animals, immunized animals, immortalized human or animalB-cells, cells transfected with DNA vectors encoding the antibody or Tcell receptor, etc. The details of the preparation of antibodies andtheir suitability for use as specific binding members are well-known tothose skilled in the art.

Of particular interest is the use of antibodies as affinity reagents.Conveniently, these antibodies are conjugated with a label for use inseparation. Labels include magnetic beads, which allow for directseparation, biotin, which can be removed with avidin or streptavidinbound to a support, fluorochromes, which can be used with a fluorescenceactivated cell sorter, or the like, to allow for ease of separation ofthe particular cell type. Fluorochromes that find use includephycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluoresceinand Texas red. Frequently each antibody is labeled with a differentfluorochrome, to permit independent sorting for each marker.

The antibodies are added to cells, and incubated for a period of timesufficient to bind the available antigens. The incubation will usuallybe at least about 5 minutes and usually less than about 30 minutes. Itis desirable to have a sufficient concentration of antibodies in thereaction mixture, such that the efficiency of the separation is notlimited by lack of antibody. The appropriate concentration is determinedby titration. The medium in which the cells are separated will be anymedium that maintains the viability of the cells. A preferred medium isphosphate buffered saline containing from 0.1 to 0.5% BSA. Various mediaare commercially available and may be used according to the nature ofthe cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank'sBasic Salt Solution (HBSS), Dulbecco's phosphate buffered saline (dPBS),RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplementedwith fetal calf serum, BSA, HSA, etc.

The cells of interest may be separated from a complex mixture of cellsby techniques that enrich for cells having the above describedcharacteristics. For isolation of cells from tissue, an appropriatesolution may be used for dispersion or suspension. Such solution willgenerally be a balanced salt solution, e.g. normal saline, PBS, Hank'sbalanced salt solution, etc., conveniently supplemented with fetal calfserum or other naturally occurring factors, in conjunction with anacceptable buffer at low concentration, generally from 5-25 mM.Convenient buffers include HEPES, phosphate buffers, lactate buffers,etc.

The separated cells may be collected in any appropriate medium thatmaintains the viability of the cells, usually having a cushion of serumat the bottom of the collection tube. Various media are commerciallyavailable and may be used according to the nature of the cells,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequentlysupplemented with fetal calf serum.

Compositions highly enriched for osteogenic progenitors are achieved inthis manner. The subject population may be at or about 50% or more ofthe cell composition, and preferably be at or about 75% or more of thecell composition, and may be 90% or more. The desired cells areidentified by their surface phenotype, by the ability to develop intobone, etc. The enriched cell population may be used immediately, or maybe frozen at liquid nitrogen temperatures and stored for long periods oftime, being thawed and capable of being reused. The cells will usuallybe stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

In one embodiment of the invention, a nucleic acid construct isintroduced into the cells of the invention. A variety of vectors areknown in the art for the delivery of sequences into a cell, includingplasmid vectors, viral vectors, and the like. In a preferred embodiment,the vector is a retroviral or lentiviral vector. For example, see Baumet al. (1996) J Hematother 5(4):323-9; Schwarzenberger et al. (1996)Blood 87:472-478; Nolta et al. (1996) P.N.A.S. 93:2414-2419; and Maze etal. (1996) P.N.A.S. 93:206-210, Mochizuki et al. (1998) J Virol72(11):8873-83. The use of adenovirus based vectors with hematopoieticcells has also been published, see Ogniben and Haas (1998) RecentResults Cancer Res 144:86-92.

Various techniques known in the art may be used to transfect the targetcells, e.g. electroporation, calcium precipitated DNA, fusion,transfection, lipofection and the like. The particular manner in whichthe DNA is introduced is not critical to the practice of the invention.

In some embodiments of the invention, a pharmaceutical composition ofthe present invention is administered to an animal to accelerate bonerepair, e.g. following an injury, in the treatment of bone disease, etc.

A cell transplant, as used herein, is the transplantation of one or morecells into a recipient body, usually for the purpose of augmentingfunction of an organ or tissue in the recipient. As used herein, arecipient is an individual to whom tissue or cells from anotherindividual (donor), commonly of the same species, has been transferred.The graft recipient and donor are generally mammals, preferably human.Laboratory animals, such as rodents, e.g. mice, rats, etc. are ofinterest for drug screening, elucidation of developmental pathways, etc.For the purposes of the invention, the cells may be allogeneic,autologous, or xenogeneic with respect to the recipient.

Where the transplantation is intended for the treatment of degenerativedisease, e.g. osteogenesis imperfecta; repair of mesenchymal tissues;etc., the cells are administered in a manner that permits them to graftor migrate to the intended tissue site and reconstitute or regeneratethe functionally deficient area.

In many clinical situations, the bone healing condition are less idealdue to decreased activity of bone forming cells, e.g. within agedpeople, following injury, in osteogenesis imperfecta, etc. A variety ofbone and cartilage disorders affect aged individuals. Such tissues arenormally regenerated by mesenchymal stem cells. Included in suchconditions is osteoarthritis. Osteoarthritis occurs in the joints of thebody as an expression of “wear-and-tear”. Thus athletes or overweightindividuals develop osteoarthritis in large joints (knees, shoulders,hips) due to loss or damage of cartilage. This hard, smooth cushion thatcovers the bony joint surfaces is composed primarily of collagen, thestructural protein in the body, which forms a mesh to give support andflexibility to the joint. When cartilage is damaged and lost, the bonesurfaces undergo abnormal changes. There is some inflammation, but notas much as is seen with other types of arthritis. Nevertheless,osteoarthritis is responsible for considerable pain and disability inolder persons.

In conditions of the aged where repair of mesenchymal tissues isdecreased, or there is a large injury to mesenchymal tissues, theosteogenic activity may be enhanced by administration of progenitorcells.

In methods of accelerating bone repair, a pharmaceutical composition ofthe present invention is administered to a patient suffering from damageto a bone, e.g. following an injury. The formulation is preferablyadministered at or near the site of injury, following damage requiringbone regeneration.

In the methods of the invention, cells to be transplanted aretransferred to a recipient in any physiologically acceptable excipientcomprising an isotonic excipient prepared under sufficiently sterileconditions for human administration. For general principles in medicinalformulation, the reader is referred to Cell Therapy: Stem CellTransplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn& W. Sheridan eds, Cambridge University Press, 1996. Choice of thecellular excipient and any accompanying elements of the composition willbe adapted in accordance with the route and device used foradministration. The cells may be introduced by injection, catheter, orthe like. The cells may be frozen at liquid nitrogen temperatures andstored for long periods of time, being capable of use on thawing. Iffrozen, the cells may be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640medium.

Expression Assays

The in vitro differentiated cells find use in the examination of geneexpression. The expressed set of genes may be compared with a variety ofcells of interest, e.g. adult osteoblasts and progenitors thereof, etc,as known in the art. For example, one could perform experiments todetermine the genes that are regulated during development.

Any suitable qualitative or quantitative methods known in the art fordetecting specific mRNAs can be used. mRNA can be detected by, forexample, hybridization to a microarray, in situ, hybridization in tissuesections, by reverse transcriptase-PCR; or in Northern, blotscontaining-poly⁺ mRNA. One of skill in the art can readily use thesemethods to determine differences in the size or amount of mRNAtranscripts between two samples. For example, the level of particularmRNAs in progenitor cells is compared with the expression of the mRNAsin a reference sample, e.g. hepatocytes, or other differentiated cells.

Hybridization to arrays may be performed, where the arrays can beproduced according to any suitable methods known in the art. Forexample, methods of producing large arrays of oligonucleotides aredescribed in U.S. Pat. Nos. 5,134,854, and 5,445,934 usinglight-directed synthesis techniques. Using a computer controlled system,a heterogeneous array of monomers is converted, through simultaneouscoupling at a number of reaction sites, into a heterogeneous array ofpolymers. Alternatively, microarrays are generated by deposition ofpre-synthesized oligonucleotides onto a solid substrate, for example asdescribed in PCT published application no. WO 95/35505.

Methods for collection of data from hybridization of samples with arraysare also well known in the art. For example, the polynucleotides of thecell samples can be generated using a detectable fluorescent label, andhybridization of the polynucleotides in the samples detected by scanningthe microarrays for the presence of the detectable label. Methods anddevices for detecting fluorescently marked targets on devices are knownin the art. Generally, such detection devices include a microscope andlight source for directing light at a substrate. A photon counterdetects fluorescence from the substrate, while an x-y translation stagevaries the location of the substrate. A confocal detection device thatcan be used in the subject methods is described in U.S. Pat. No.5,631,734. A scanning laser microscope is described in Shalon et al.,Genome Res. (1996) 6:639. A scan, using the appropriate excitation line,is performed for each fluorophore used. The digital images generatedfrom the scan are then combined for subsequent analysis. For anyparticular array element, the ratio of the fluorescent signal from onesample is compared to the fluorescent signal from another sample, andthe relative signal intensity determined.

In other screening methods, the test sample is assayed at the proteinlevel. Diagnosis can be accomplished using any of a number of methods todetermine the absence or presence or altered amounts of a differentiallyexpressed polypeptide in the test sample. For example, detection canutilize staining of cells or histological sections (e.g., from a biopsysample) with labeled antibodies, performed in accordance withconventional methods. Cells can be permeabilized to stain cytoplasmicmolecules. In general, antibodies that specifically bind adifferentially expressed polypeptide of the invention are added to asample, and incubated for a period of time sufficient to allow bindingto the epitope, usually at least about 10 minutes. The antibody can bedetectably labeled for direct detection (e.g., using radioisotopes,enzymes, fluorescers, chemiluminescers, and the like), or can be used inconjunction with a second stage antibody or reagent to detect binding(e.g., biotin with horseradish peroxidase-conjugated avidin, a secondaryantibody conjugated to a fluorescent compound, e.g. fluorescein,rhodamine, Texas red, etc.). The absence or presence of antibody bindingcan be determined by various methods, including flow cytometry ofdissociated cells, microscopy, radiography, scintillation counting, etc.Any suitable alternative methods of qualitative or quantitativedetection of levels or amounts of differentially expressed polypeptidecan be used, for example ELISA, western blot, immunoprecipitation,radioimmunoassay, etc.

Screening Assays

The subject cells are useful for in vitro assays and screening to detectagents that affect osteoblasts and cells in the osteogenic lineage. Awide variety of assays may be used for this purpose, includingtoxicology testing, immunoassays for protein binding; determination ofcell growth, differentiation and functional activity; production ofhormones; and the like.

In screening assays for biologically active agents, drugs, etc., thesubject cells, usually a culture comprising the subject cells, iscontacted with the agent of interest, and the effect of the agentassessed by monitoring output parameters, such as expression of markers,cell viability, and the like. The cells are typically in vitro culturedcells, and may include clonal cultures: e.g. split into independentcultures and grown under distinct conditions, for example with orwithout virus; in the presence or absence of other cytokines orcombinations thereof. The manner in which cells respond to an agent,particularly a pharmacologic agent, including the timing of responses,is an important reflection of the physiologic state of the cell.

Parameters are quantifiable components of cells, particularly componentsthat can be accurately measured, desirably in a high throughput system.A parameter can be any cell component or cell product including cellsurface determinant, receptor, protein or conformational orposttranslational modification thereof, lipid, carbohydrate, organic orinorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. While mostparameters will provide a quantitative readout, in some instances asemi-quantitative or qualitative result will be acceptable. Readouts mayinclude a single determined value, or may include mean, median value orthe variance, etc. Characteristically a range of parameter readoutvalues will be obtained for each parameter from a multiplicity of thesame assays. Variability is expected and a range of values for each ofthe set of test parameters will be obtained using standard statisticalmethods with a common statistical method used to provide single values.

Agents of interest for screening include known and unknown compoundsthat encompass numerous chemical classes, primarily organic molecules,which may include organometallic molecules, inorganic molecules, geneticsequences, etc.

In addition to complex biological agents, candidate agents includeorganic molecules comprising functional groups necessary for structuralinteractions, particularly hydrogen bonding, and typically include atleast an amine, carbonyl, hydroxyl or carboxyl group, frequently atleast two of the functional chemical groups. The candidate agents oftencomprise cyclical carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more of the abovefunctional groups. Candidate agents are also found among biomolecules,including peptides, polynucleotides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Included are pharmacologically active drugs, genetically activemolecules, etc. Compounds of interest include chemotherapeutic agents,hormones or hormone antagonists; etc. Exemplary of pharmaceutical agentssuitable for this invention are those described in, “The PharmacologicalBasis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y.,(1996), Ninth edition, under the sections: Water, Salts and Ions; DrugsAffecting Renal Function and Electrolyte Metabolism; Drugs AffectingGastrointestinal Function; Chemotherapy of Microbial Diseases;Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Formingorgans; Hormones and Hormone Antagonists; Vitamins, Dermatology; andToxicology, all incorporated herein by reference. Also included aretoxins, and biological and chemical warfare agents, for example seeSomani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, NewYork, 1992).

Test compounds include all of the classes of molecules described above,and may further comprise samples of unknown content. Of interest arecomplex mixtures of naturally occurring compounds derived from naturalsources such as plants. While many samples will comprise compounds insolution, solid samples that can be dissolved in a suitable solvent mayalso be assayed. Samples of interest include environmental samples, e.g.ground water, sea water, mining waste, etc.; biological samples, e.g.lysates prepared from crops, tissue samples, etc.; manufacturingsamples, e.g. time course during preparation of pharmaceuticals; as wellas libraries of compounds prepared for analysis; and the like. Samplesof interest include compounds being assessed for potential therapeuticvalue, i.e. drug candidates.

The term “samples” also includes the fluids described above to whichadditional components have been added, for example components thataffect the ionic strength, pH, total protein concentration, etc. Inaddition, the samples may be treated to achieve at least partialfractionation or concentration. Biological samples may be stored if careis taken to reduce degradation of the compound, e.g. under nitrogen,frozen, or a combination thereof. The volume of sample used issufficient to allow for measurable detection, usually from about 0.1 μlto 1 ml of a biological sample is sufficient.

Compounds, including candidate agents, are obtained from a wide varietyof sources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds, including biomolecules,including expression of randomized oligonucleotides and oligopeptides.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or readily produced.Additionally natural or synthetically produced libraries and compoundsare readily modified through conventional chemical, physical andbiochemical means, and may be used to produce combinatorial libraries.Known pharmacological agents may be subjected to directed or randomchemical modifications, such as acylation, alkylation, esterification,amidification, etc. to produce structural analogs.

Agents are screened for biological activity by adding the agent to atleast one and usually a plurality of cell samples, usually inconjunction with cells lacking the agent. The change in parameters inresponse to the agent is measured, and the result evaluated bycomparison to reference cultures, e.g. in the presence and absence ofthe agent, obtained with other agents, etc.

The agents are conveniently added in solution, or readily soluble form,to the medium of cells in culture. The agents may be added in aflow-through system, as a stream, intermittent or continuous, oralternatively, adding a bolus of the compound, singly or incrementally,to an otherwise static solution. In a flow-through system, two fluidsare used, where one is a physiologically neutral solution, and the otheris the same solution with the test compound added. The first fluid ispassed over the cells, followed by the second. In a single solutionmethod, a bolus of the test compound is added to the volume of mediumsurrounding the cells. The overall concentrations of the components ofthe culture medium should not change significantly with the addition ofthe bolus, or between the two solutions in a flow through method.

Preferred agent formulations do not include additional components, suchas preservatives, that may have a significant effect on the overallformulation. Thus preferred formulations consist essentially of abiologically active compound and a physiologically acceptable carrier,e.g. water, ethanol, DMSO, etc. However, if a compound is liquid withouta solvent, the formulation may consist essentially of the compounditself.

A plurality of assays may be run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in the phenotype.

Various methods can be utilized for quantifying the presence of theselected parameters, or markers. For measuring the amount of a moleculethat is present, a convenient method is to label a molecule with adetectable moiety, which may be fluorescent, luminescent, radioactive,enzymatically-active, etc., particularly a molecule specific for bindingto the parameter with high affinity Fluorescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g. by expressing them asgreen fluorescent protein chimeras inside cells (for a review see Joneset el. (1999) Trends Biotechnol. 17(12):477-81). Thus, antibodies can begenetically modified to provide a fluorescent dye as part of theirstructure. Depending upon the label chosen, parameters may be measuredusing other than fluorescent labels, using such immunoassay techniquesas radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA),homogeneous enzyme immunoassays, and related non-enzymatic techniques.The quantitation of nucleic acids, especially messenger RNAs, is also ofinterest as a parameter. These can be measured by hybridizationtechniques that depend on the sequence of nucleic acid nucleotides.Techniques include polymerase chain reaction methods as well as genearray techniques. See Current Protocols in Molecular Biology, Ausubel etal., eds, John Wiley & Sons, New York, N.Y., 2000; Freeman et al. (1999)Biotechniques 26(1):112-225; Kawamoto et al. (1999) Genome Res9(12):1305-12; and Chen et al. (1998) Genomics 51(3):313-24, forexamples.

Kits

The culture systems of the invention are optionally packaged in asuitable container with written instructions for a desired purpose. Suchformulations may comprise medium, growth factors, etc., in a formsuitable for combining with cells prior to culture.

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. General methodsin molecular and cellular biochemistry can be found in such standardtextbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrooket al., Harbor Laboratory Press 2001); Short Protocols in MolecularBiology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); ProteinMethods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors forGene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors(Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual(I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture:Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley &Sons 1998). Reagents, cloning vectors, and kits for genetic manipulationreferred to in this disclosure are available from commercial vendorssuch as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

Other features and advantages of the invention will be apparent from thedescription of the preferred embodiments, and from the claims. Thefollowing examples are offered by way of illustration and not by way oflimitation.

EXPERIMENTAL Material and Methods

Isolation and Culture of Mouse BMSCs. Isolation of MSCs from mouse bonemarrow was according to Peister et al. (2004) Blood 103(5):1662-8.Female and male BALB/c mice 3 weeks old were individually euthanizedusing CO₂. The femurs and tibiae were removed, cleaned of all connectivetissue, and placed on ice in 2 mL complete isolation media (CIM). CIMconsisted of α-MEM (Invitrogen, Carlsbad, Calif.) supplemented with 20%fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, Ga.), 100 U/mLpenicillin (Invitrogen), 100 μg/mL streptomycin (Invitrogen), and 12 μML-glutamine (Invitrogen). The ends of each tibia and femur were clippedto expose the marrow, flushed out using a 20 gauge needle, andcentrifuged for 1 minute at 1200 rpm. The pellet was resuspended in 1 mLCIM with a micropipette. The cells from 2 mice were plated in 10 mL CIMin a 100 mm culture dish.

After 24 hours, nonadherent cells were removed by washing withphosphate-buffered saline (PBS), and 10 mL fresh CIM was added. Theadherent cells (passage 0) were washed, and media changed with fresh CIMevery 3 days for a period of 1 week. After 1 week, the cells were washedwith PBS and detached by incubation in 1 mL 0.25% trypsin/1 mMethylenediaminetetraacetic acid (EDTA; Invitrogen) for 2 minutes at 37°C. The cells that did not lift in 2 minutes were discarded. The trypsinwas neutralized by the addition of 5 mL CIM, and all the cells(passage 1) from one dish were replated in 10 mL CIM in a 100 mm culturedish. The CIM was replaced every 3 days. After 1 week, the cells werelifted by incubating with trypsin/EDTA for 2 minutes at 37° C. The cells(passage 2) were then expanded by plating at 50 cells/cm² in completeexpansion media (CEM) consisting of Iscove modified Dulbecco medium(IMDM; Invitrogen) supplemented with 9% FBS, 9% Horse serum(Invitrogen), 100 U/mL penicillin, 100 μg/mL streptomycin, and 12 μML-glutamine. The CEM was replaced every 3 to 4 days. After 1 to 2 weeks,the cells (passage 3) were lifted by incubation with trypsin/EDTA for 2minutes at 37° C. The passage 3 cells were either frozen or expandedfurther by plating at 50 cells/cm² and incubation in CEM.

Osteogenic and Adipogenic Differentiation of BMSCs. Osteogenicdifferentiation was induced by culturing cells in osteogenic medium(OS-medium, Cell Applications, Inc. CA) for 7 days. Adipogenicdifferentiation was induced by culturing cells in osteogenic medium(AD-medium, Cell Applications, Inc. CA) for 7 days.

After osteogenic induction cells were stained with alizarin red S (AR-S,Sigma, San Louis, Ind.). Cells were rinsed in PBS and incubated with 40mM AR-S (pH 4.2) with rotation for 10 min, then rinsed 5 times withwater followed by a 15 min wash with PBS with rotation to reducenonspecific AR-S staining. The stained nodules were visualized using alight field microscope. Cells were washed with PBS and fixed with 10%formalin for 20 min. Cells were then washed twice with PBS and once with60% isopropyl alcohol, and stained with Oil red O solution (Sigma). Thestained nodules were observed through the microscope.

For detection of bone mineralization, we followed previously publishedmethods were utilized. After culturing MSCs for 14 days, the media wasaspirated and cells stained with alkaline phosphatase (StemTAG, CellBiolabs, Inc. CA) according to the manufacturer's protocol and observedunder light microscopy.

An alkaline phosphatase (ALP) assay was performed in cell layers bycalorimetric assay of enzyme activity using an Alkaline phosphatase kit(Cell Biolab, Inc.), following the manufacturer' instructions. Celllayers were washed 3 times with PBS, then total proteins extracted usingthe Protein Extract Reagents kit (Pierce, Rockford, Ill.), followed bycentrifugation to remove cellular debris. Fifty μL of lysate was thenmixed with 50 μL of the freshly prepared calorimetric substratepara-nitrophenyl phosphate, and incubated at 37° C. for 30 min. Theenzymatic reaction was stopped by adding 50 μL of 0.2 N NaOH. Theoptical density of the yellow product para-nitrophenol was determined bya HTS 7000 Plus Bio Assay reader (PE, USA) at 405 nm. Proteinconcentration of the cell lysates was measured with a BCA Protein AssayKit (Pierce) and ALP activity was then expressed as O.D.405 nm/mg ofprotein.

Cytokine induction studies culture. At passage 5 or 6, MSC were placedin 60 mm culture dishes at a density of 2.5×10⁵ cells/well in completeexpansion media (CEM). When cells reached 80-90% confluence, growthmedium was supplemented with 10 ng/ml of TGF-β1 for 14 days. Media waschanged every other day. Total RNA was isolated after 14 days usingTRIzol (Invitrogen). RNA was dissolved in ddH₂O and stored at −80° C.The yield of RNA was determined by measuring absorbance at 260 nm.

Real-time RT-PCR. Reverse transcriptase (RT) reactions were annealed at24° C. for 10 min, followed by first-strand cDNA synthesis at 48° C. for1 hr and heat inactivation at 95° C. for 5 min. The resulting cDNA wasstored at −20° C. until assayed by real-time PCR.

Real-time PCR analysis was performed using SYBR Green PCR core reagents(Applied Biosystems, Foster City, Calif.) following the manufacturer'sprotocol on an ABI Prism 7700 Sequence Detection System. All primers(see Table 1) were designed using the Primer3 program (WhiteheadInstitute, Cambridge, Mass.). Briefly, the real-time PCR reactions wereperformed with 10 μl each of SYBR Green kit master mix, and forward andreverse primers. Six μL of cDNA sample was added to 54 μL of finalreaction mixture. The 384-well real-time PCR format included six 2-folddilutions in triplicate of the plasmid DNA standards. The wells of theplate were sealed with optical adhesive covers (Bio-Rad Laboratories,Hercules, Calif.) and centrifuged at low speed (300×g, 5 min) to ensurecomplete mixing. Each sample was analyzed at least in triplicate.

Standard PCR involved activation of DNA polymerase followed by 40 cyclesof denaturation at 94° C. for 30 s, annealing at 60° C. for 30 s, andextension at 72° C. for 30 s. The PCR threshold cycle number (C_(T)) foreach sample was calculated at the point where the fluorescence exceededthe threshold limit. The threshold limit was fixed along the linearlogarithmic phase of the fluorescence curves at 10 to 20 standarddeviations (SDs) above the average background fluorescence. Relativeexpression levels were calculated using the standard curve methodrecommended by Applied Biosystems.

Statistical analysis. We performed three or more independent sets of theexperiments, and each experiment was run at least three times. Data wereshown as means ±SD and analyzed by a paired analysis of variance. Pvalues were described in figures and P<0.05 was considered asstatistically significant.

Results

Bone Marrow-Derived MSC Isolation and Characterization. Marrow wasisolated from the long bones of mice and seeded in culture dishes. Cellsat passage 5-7 were used in the experiments. FIG. 1A shows the classicalspindle morphology characteristic of BMSCs. Stem cell antigen-1 (Sca-1)is an 18-kDa mouse glycosyl phosphatidylinositol-anchored cell surfaceprotein and has been used routinely in combination with negativeselection against mature markers for enrichment of stem and progenitorcells (Holmes and Stanford (2007). Stem Cells 25(6):1339-47). In orderto confirm if Sca-1 was expressed on BMSCs, immunocytochemistry wasperformed. FIG. 1C demonstrates that almost all BMSCs were found toexpress Sca-1 at the cell surface. In contrast, no positive staining wasobserved with BMSCs exposed to an isotype control IgG (FIG. 1B).

In order to identify if multipotency was maintained, BMSCs were culturedin osteogenic or adipogenic induction medium for 7 days. Cells were thenwashed with PBS and stained with alizarin red or red oil O. FIG. 2Areveals ˜that 80% of cells in the osteogenic medium were positive foralizarin red staining indicating osteogenic differentiation, whereasBMSCs which were cultured in IMDM medium were negative (FIG. 2B).Similarly, BMSCs cultured in adipogenic medium stained positively withred oil O indicating adipogenic differentiation (FIG. 2C). No stainingwas observed with BMSCs cultured in basic control medium (FIG. 2D).

The effect of TGF-β1 on differentiation of BMSCs. TGF-β has been shownto stimulate chondrocytic differentiation in vitro, but whether itmodulates osteoblastic differentiation remains unclear. In order toclarify this question, BMSCs were cultured in IMDM media with or withoutTGF-β1 for 14 days. mRNA was then isolated and RT-PCR was perfused toexamine the expression of stem cell markers in BMSCs. FIG. 3 shows thatTGF-β1 reduced expression of abcg2 (0.60±0.00), nanos3 (0.28±0.03), Oct4(0.16±0.03), and stella (0.22±0.05), indicating downregulation ofself-renewal pathways. FIG. 3 also shows that the expression ofosteogenic transcript factors, TAZ and runx2, was increased 2.9±0.3 and2.6±0.7 fold, respectively, in the TGF-β1 treated group relative tountreated controls. Similarly, the expression of osteoblast markers,osteopontin and collagen I was enhanced 6.0±1.4 and 4.1±1.7 fold in thetreated group (FIG. 3). In contrast, mRNA levels of adipocyte markers,PPARγ2 and adipsin, were reduced to 0.80±0.14 and 0.15±0.06,respectively, in the TGF-β1 treated group (FIG. 3). Surprisingly, mRNAlevels of chondrocyte markers (collagen types II, IX and aggrecan) werereduced 50% relative to controls.

To further confirm these findings, the expression and activity ofalkaline phosphatase (AP), a marker of bone differentiation, wasinvestigated in TGF-β1 treated BMSCs. FIG. 4A shows that the AP stainingincreased significantly after days or weeks of TGF-β1 treatment comparedto untreated cells. Furthermore, AP activity was dramatically increasedto 368±100 nmol N—PPN/min/mg in TGF-β1 treated BMSCs, compared to 25±2nmol N—PPN/min/mg in untreated cells (FIG. 4B).

To better understand the effects of TGF-β1 on BMSC osteogenicdifferentiation, we examined for expression of downstream effectors ofTGF-β1 signaling by RT-PCR. FIG. 5 shows that mRNA levels of smad 2 andsmad 3 were increased 1.9±0.6 and 1.6±0.6 fold, respectively, afterBMSCs were treated with TGF-β1. Furthermore, the expression of TGF-β1was increased 4.5±0.6 fold.

To our knowledge, this is the first study to report on the osteogenicpotential of recombinant TGF-β1 in bone marrow-derived MSCs in vitro.Our data confirm that TGF-β1 not only significantly enhances cellproliferation, but also stimulates osteogenesis in BMSCs as assessed byimmunohistochemical, biochemical, and gene expression studies performedherein. Furthermore, this study also demonstrates the ability of TGF-β1to significantly promote TAZ and runx2 mRNA levels in BMSCs in vitro

Our results indicate that a more physiological agonist such as TGF-β1may possess therapeutic potential as an osteogenic induction factor,compared to the traditional induction protocols involving a combinationof dexamethasone, β-glycerophosphate and ascorbic acid. Previous studieshave shown that osteogenic factors such as BMP-2 and BMP-4 are absentduring the whole healing process in humans (Zimmermann et al. (2005)Bone 36(5): 779-85), while TGF-β1 was shown to rise in peripheral blooddramatically at two weeks post-trauma and for at least an additional sixmonths. Therefore, it is possible that TGF-β1 may play a role in bonefracture healing and that exogenous supplementation may overcome otherphysiological limitations such as enzymatic degradation or cross-talkfrom other signaling pathways induced post-injury.

Several studies have been shown that TGF-β1 stimulated chondrocyticdifferentiation in vitro (Han et al. (2005) J Cell Biochem 95(4):750-62;Mehlhorn et al. (2006) Tissue Eng 12(10):2853-62). However, in oursystem TGF-β1 decreased the expression of chondrogenic markers, such ascollagen II, collagen IX and aggrecan at 14 days. The explanation may bethat TGF-β1 acts sequentially to drive successive stages ofdifferentiation during cartilage and bone development.

Large bodies of evidence indicate that Runx2 is an essentialtranscription factor for bone formation (Ducy et al. (2000) Science289(5484):1501-4; Yamaguchi et al. (2000) Endocr Rev 21(4):393-411).However, it has been suggested that other transcriptional regulators arecooperatively involved in the osteogenic action of Runx-2 because thetranscriptional activity of Runx2 itself is relatively weak Kanno e al.(1998) Mol Cell Biol 18(5):2444-54) TAZ, a transcriptional coactivatorwith PDZ-binding motif, interacts with a variety of transcriptionfactors and exhibits transcriptional regulatory functions. TAZ isbelieved to regulate gene expression during embryogenesis anddevelopment of bone, muscle, fat, lung, heart, and limbs. Furthermore, arecent study indicates that TAZ acts as a transcriptional regulator forthe differentiation of MSCs into osteoblast cells (Hong et al, supra).In this case, TAZ functions as a coactivator of Runx2 and acts as acorepressor of PPARγ, which is a master regulator of adipocytedifferentiation. These reports reveal that TAZ plays an important rolein MSC differentiation; however, the exact upstream triggers of thispathway were not elucidated. Our results showed that activation ofTGF-β-Smad signaling promoted TAZ and Runx2 expression and osteoblastdifferentiation. Transcripts of several components of TGF-β1-Smadpathway were up-regulated during differentiation: TGF-β1, Samd-2,Samd-3, and Act-2a on day 14. This study is the first to reveal thatTGF-β1 enhance the TAZ mRNA level mediated by the TGF-β-Smad signalingpathway.

In conclusion, the identification of functional roles of TGF-β1 inosteoblast differentiation provides a novel insight into understandingthe molecular mechanism of the commitment of mesenchymal stem cells inbone marrow and may allow as to develop new therapeutic for bone diseasesuch as osteoporosis, bone fracture.

TABLE 1 Primers used for real-time polymerase chain reaction SequenceGene marker identifier Primers sequence Runx2 SEQ ID NO:1 (5′-3′) CCCAGC CAC CTT TAC CTA CA SEQ ID NO:2 (3′-5′) TAT GGA GTG CTG CTG GTC TGTAZ SEQ ID NO:3 (5′-3′) TCC CCA CAA CTC CAG AAG AC SEQ ID NO:4 (3′-5′)CAA AGT CCC GAG GTC AAC AT Osteopontin SEQ ID NO:5 (5′-3′) CAC TCC AATCGT CCC TAC SEQ ID NO:6 (5′-3′) AGA CTC ACC GCT CTT CAT Osteocalcin SEQID NO:7 (5′-3′) AAG CCC AGC GAC TCT GAG TC SEQ ID NO:8 (3′-5′) GCT CCAAGT CCA TTG TTG AGG Collagen I SEQ ID NO:9 (5′-3′) ATG GAG ACA GGT CAGACC TGT GT SEQ ID NO:10 (3′-5′) TCG GTC ATG CTC TCT CCA AAC Adipsin SEQID NO:11 (5′-3′) GCT ATC CCA GAA TGC CTC GTT SEQ ID NO:12 (3′-5′) GCGTGC CGG GTT CCA PPARgamma SEQ ID NO:13 (5′-3′) AAC CAT TGG GTC AGC TCTTG SEQ ID NO:14 (5′-3′) GAT GGA AGA CCA CTC GCA TT TGF-β1 SEQ ID NO:15(5′-3′) AAC AAT TCC TGG CGT TAC CTT SEQ ID NO:16 (3′-5′) ATT CCG TCT CCTTGG TTC AG Smad-2 SEQ ID NO:17 (5′-3′) GGA AAG GGT TGC CAC ATG TTA T SEQID NO:18 (3′-5′) GCA GTT TTC GAT TGC CTT GAG Smad-3 SEQ ID NO:19 (5′-3′)GGG CCT ACT GTC CAA TGT CAA SEQ ID NO:20 (3′-5′) CGC ACA CCT CTC CCA ATGT Smad-4 SEQ ID NO:21 (5′-3′) CGC TTT TGC TTG GGT CAA CT SEQ ID NO:22(5′-3′) TGT GCA ACC TCG CTC TCT CA

1. A method for obtaining osteogenic progenitor cells, the methodcomprising: culturing isolated mesenchymal stem cells in vitro in thepresence of a TGF-β factor at a concentration of from about 100 pg/ml toabout 100 ng/ml for a period of time sufficient to induce osteogenicdifferentiation.
 2. The method according to claim 1, wherein the TGF-βfactor is present at around about 10 ng/ml.
 3. The method of claim 1,wherein the TGF-β factor is TGF-β1.
 4. The method of claim 1, whereinthe TGF-β factor is TGF-β3.
 5. The method of claim 1, further comprisingthe step of isolating osteogenic progenitor cells from the culture. 6.The method of claim 1, wherein TAZ activity is upregulated.
 7. Themethod according to claim 1, wherein the mesenchymal stem cells areisolated from adipose tissue.
 8. The method according to claim 1,wherein the mesenchymal stem cells are isolated from bone marrow.